DESCRIPTION (adapted from the investigator's application): Ca2+ entry through voltage sensitive Ca2+ channels is the trigger for exocytosis in excitable cells. Recent evidence suggests that spatially localized Ca2+ gradients are important for regulating exocytosis. The goal of this proposal is to combine the pulsed laser Ca2+ imaging and confocal spot detection of Ca2+ transients with state-of-the-art methods for measuring exocytosis from single cells (patch clamp and voltametric measurements) in order to investigate the regulation of exocytosis by Ca2+ gradients in adrenal chromaffin cells. The experiments are designed to address the following questions. (1) What are the physiological mechanisms that control the development and extent of Ca2+ gradients at exocytotic sites? (2) What is the relationship between the Ca2+ gradients and exocytosis during depolarizing stimuli? (3) How do trains of short depolarizations, which are highly effective stimuli for exocytosis, alter the Ca2+ gradients and recruit secretory vesicles? (4) What are the roles of Ca2+ channel localization and Ca2+ buffers in Ca2+ signal localization? (5) Do the exocytotic sites behave as functional 'active zones'? And (6) what are the roles of Ca2+ gradients and GTP-binding proteins in recruitment of secretory vesicles to the exocytotic sites?